WO1997022942A1 - Procede de deconvolution servant a effectuer l'analyse de donnees obtenues au moyen de processus de separation analytique - Google Patents
Procede de deconvolution servant a effectuer l'analyse de donnees obtenues au moyen de processus de separation analytique Download PDFInfo
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- WO1997022942A1 WO1997022942A1 PCT/US1996/019242 US9619242W WO9722942A1 WO 1997022942 A1 WO1997022942 A1 WO 1997022942A1 US 9619242 W US9619242 W US 9619242W WO 9722942 A1 WO9722942 A1 WO 9722942A1
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F18/00—Pattern recognition
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8603—Signal analysis with integration or differentiation
- G01N30/8617—Filtering, e.g. Fourier filtering
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F2218/00—Aspects of pattern recognition specially adapted for signal processing
- G06F2218/02—Preprocessing
Definitions
- This invention relates to the analysis of data resulting from an analytical separation process using an improved deconvolution method
- Analytical separation process separate individual components of a multicomponent mixture into individual sample zones based on the different migration 5 rate of each component
- Examples of such separation process include liquid chromatography, gas chromatography, electrophoresis, centrifugation, staged extraction, adsorption, and the like
- DNA sequencing relies on the electrophoretic separation of polynucleotide fragments up to 1000 nucleotides in length which differ in size by only a single nucleotide
- Data resulting from analytical separation process is typically in the form of sample quantity as a function of time and/or position
- the data is in the form of concentration as a function of time
- the data is in the form of sample quantity as a function of position
- a problem which a ⁇ ses in the interpretation of such data is the identification of individual sample zones from among a plurality of zones which have not been completely resolved, i e , overlapping sample zones See FIG 1 Lack of resolution is caused by the finite width of a sample zone due to the dispersive effects of diffusion, interfacial mass transfer, thermal profiles, sample injection, and other like effects This problem is particularly acute when automatic data analysis is utilized, e , automatic base calling of DNA sequences, where hundreds of overlapping zones must be accurately resolved
- a measured signal is considered to be a convolution of two separate signal functions— a "true" signal function representing the location of a hypothetical zone having zero width, and a point-spread-function (PSF) representing the non-zero width of each sample zone
- the deconvolution process generally is implemented by taking the Fourier transform (FT) of the signal and a PSF, dividing the transformed signal by the transformed PSF, and performing an inverse Fourier transform of the result
- FT Fourier transform
- a filtering operation may applied prior to taking the inverse Fourier transform to reduce the effect of noise on the deconvolved data
- blind deconvolution methods applied to data resulting from analytical separations use a "blind" deconvolution method with homomorphic filtering, e g , U S Patent No 5,273,632, inco ⁇ orated herein by reference in its entirety
- Blind deconvolution methods utilize a constant PSF which is chosen without any reference
- the present invention is directed toward our discovery of an improved signal processing method in a signal processor for performing the deconvolution of a signal resulting from an analytical separation process, e g , electrophoresis, HPLC or other like processes
- the method of the invention uses priori knowledge of the nature of the signal and/or adaptive parameter estimation techniques to tune a PSF used to estimate the width of a sample zone
- the method has particular application in the area of automated DNA sequencing
- An object of our invention is to provide an improved signal processing method for identifying individual sample zones in a signal containing multiple overlapping sample zones wherein a highly discriminatory filter can be applied to the transformed signal, e g , a Werner filter
- Another object of our invention is to provide an improved signal processing method for identifying individual sample zones in a signal containing multiple overlapping sample zones wherein a PSF is determined using an application-specific model of the PSF which takes into account the variation of the PSF as a function of time and/or position in the signal
- Yet another object of our invention is to provide an improved signal processing method for identifying individual sample zones in a signal containing multiple overlapping sample zones wherein the value of the PSF is determined using adaptive parameter estimation techniques
- Another object of our invention is to provide an improved signal processing method which facilitates the automatic base-calling of DNA sequences
- an improved signal processing method in a signal processor comprising the following steps A signal D(t) representing a plurality of partially separated sample zones is measured in a data window and received bv the processor A pomt-spread- function of the signal is then determined for that data window, P(t), and the Fourier transform of the signal and the point-spread-function is taken Next a noise component n of the signal is determined and the value of a result signal A(f) is calculated, where
- the point-spread function is a Gaussian function having a standard deviation ⁇ , where ⁇ is determined using an ⁇ tracker including constant terms ⁇ and ⁇ , where ⁇ is between 0 2 and 0.8 and
- the point-spread function is a Gaussian function having a standard deviation ⁇ , where ⁇ is determined using the function where a and b are constants
- a plurality of possible point-spread- functions of the signal are determined Then, the Fourier transform of each of the point-spread-functions is taken along with the Fourier transform of the signal Next, an estimate of a noise component of the signal is determined and the value of a result signal A(f) is calculated for each point-spread-function, where
- variables D(f), P(f), P*(f) ⁇ and n are as defined above
- the inverse Fourier transform of the result signal A(f) is taken for each value of the point-spread function Then, the value of the largest point-spread-function which provides a non-negative result signal in the data window is determined, and the associated value of A(t) is reported.
- a program storage device readable by a machine, tangibly embodying a program of instructions executable by a machine to perform the method steps of the first or the second aspects of the invention is provided.
- a signal processor adapted to carry out the method steps of the first or the second aspects of the invention is provided.
- FIG 1 is a schematic representation of overlapping sample zones in a signal
- FIG 2 is a flow chart of generally describing the signal processor of the present invention
- FIG 3 depicts a normal or Gaussian distribution
- FIG 4 is a flow chart of steps generally describing a preferred ⁇ -tracker utilized in the signal processor of the present invention
- FIGS 5 A, 5B, and 5C show alternative methods for indexing a data window utilized in the signal processor of the invention
- FIGS 6A and 6B show DNA sequencing data obtained early in a run both before (top) and after (bottom) processing by the signal processor of the invention
- FIGS. 7 A and 7B show DNA sequencing data obtained in the middle of a run both before (top) and after (bottom) processing by the signal processor of the invention
- FIGS. 8 A and 8B show DNA sequencing data obtained towards the end of a run both before (top) and after (bottom) processing by the signal processor of the invention DESCRIPTION OF THE PREFERRED EMBODIMENTS
- the present invention is directed towards an improved signal processing method in a signal processor useful for identifying individual sample zones, or "peaks", present in a signal comp ⁇ sing a plurality of overlapping sample zones, such signal being generated by a detector monito ⁇ ng an analytical separation process, e g , chromatography, electrophoresis, cent ⁇ fugation, staged extraction adsorption, and the like More specifically, the invention is directed toward an improved signal processing method in a signal processor for deconvolving such a signal The invention is particularly well adapted for detecting individual electrophoretic sample zones in a signal containing multiple overlapping sample zones resulting from an automated DNA sequencing process
- the method of the present invention is earned out as follows (i) the Fourier transform of a signal in a data window is computed, (u) a point-spread function of the signal is determined, (in) the Fourier transform of the point-spread function is taken, (iv) a noise component of the signal is determined, (v) the value of a deconvolved result signal A(f) is determined by the formula
- FIG. 2 shows a representation of the signal processing system of the present invention.
- the signal processor of the invention receives a signal generated by a detector which monitors the results of an analytical separation process, e.g., an electrophoretic separation.
- detectors may be "real-time" detectors which monitor the output of a separation during the separation, e.g., a HPLC chromatograph or a real-time electrophoresis scanner.
- the signal is generated by an electrophoresis scanner useful for separating and detecting the products of a DNA sequencing reaction, e.g., by Sanger-type sequencing, e.g., U.S. Patent Nos. 4,81 1 ,218, 4,879,012, 4,832,815, 4,675,095, and 5,274,240, each of which is incorporated by reference herein in its entirety.
- the detector may be a "snap-shot" detector which analyzes the results of a separation after the separation is completed, e.g., an autoradiographic or fluorescent scanner, e.g., U.S. Patent No. 5,091 ,652, which is inco ⁇ orated by reference herein in its entirety.
- Such detectors may rely on any detectable signal which can be associated with a sample component.
- Exemplary detectors include detectors based on changes in fluorescence, absorbance, index of refraction, radioactivity, or the like.
- the signal-to-noise ratio (S/N) of the signal is greater than 10 dB.
- the sampling frequency of the signal is chosen to satisfy the Nyquist sampling theorem, i.e., the sampling frequency is at least twice the frequency of the highest frequency in the signal.
- the signals are converted to digital form prior to processing using standard A/D conversion apparatus.
- PSF point-spread function
- the model can be in the form of a function, or as a set of discrete values
- Exemplary PSFs include functions such as cos, sin, cos 2 , Lorentzean, second order parabolic , Gaussian, or any other like function or combination of functions
- the PSF is a Gaussian function or a group of multiple Gaussian functions
- the ability of a deconvolution method to identify overlapping sample zones is directly related to how well the PSF approximates the signal, or how well "tuned” the PSF is to a particular signal If the PSF is not properly tuned, the deconvolution process will not faithfully recover the "true" signal, and may result in the creation of spurious peaks and/or the loss of actual peaks Given a Gaussian PSF, the parameter s is used to tune the PSF to the signal
- the value of ⁇ at a particular location in a signal derived from a DNA sequencing electrophoresis experiment is computed using an empirical function which describes the change in s as a function of time or iocation
- a function which describes how ⁇ changes with base number rather than a using a constant value for ⁇
- the PSF is better tuned to the signal throughout the data
- a and b are constants which are chosen by fitting experimental data Values of a and b are dependent on the conditions used for the electrophoretic separation, e g., electrical field strength, gel concentration, buffer composition, temperature, and other like parameters which affect electrophoretic separations
- a is preferably about 8 61 s 2
- the value of ⁇ is determined using adaptive parameter estimation, where, as used herein, the terms “adaptive parameter estimation” or “adaptive tuning” refer to a process whereby the value of a parameter is determined with reference to previous values of that parameter in the same signal Adaptive tuning has the advantage that the tuning algorithm can dynamically adapt to a signal in which the value of ⁇ changes in an unpredictable manner.
- the adaptive tuning is accomplished using an ⁇ tracker, e.g , Multiple-Target Tracking with Radar Applications, S.S Blackman, Artech House, Inc (1986)
- the ⁇ tracker is preferred because it is computationally efficient and provides sufficiently accurate tracking
- the term " ⁇ tracker” refers to a method for updating the value of a variable in a data window based on (i) an initial value of a dependent variable, (ii) a present value of a dependent variable; and (iii) a present value of the first derivative of the dependent variable with respect to a relevant independent variable, e.g , time or position
- the term “data window” refers to a portion of the signal to which one cycle of the ab tracker is applied See FIGS 5A-C The size of the data window is chosen such that the value of ⁇ within the window is essentially constant
- values are measured directly from a data window in which such values can be accurately measured
- the earlv bases, l e between 20 and 100 nucleotides, are typically completely resolved
- values of s can be easily measured using any peak-identification method, e g , the 0-cross ⁇ ng method (The 0-cross ⁇ ng method will be desc ⁇ bed hereinafter )
- the data window is indexed from the initial k window to the k+1 window.
- the k+ 1 window can be (l) immediately adjacent to the k window, FIG 5A, (n) partially overlap the k window, FIG 5B, or (in) be distant from the k window with uninterrogated data located between the k and k+1 windows, FIG 5C
- the choice of indexing method depends on several factors including (I) the magnitude of d ⁇ /dt, (u) the required accuracy, and (in) the required computational speed
- T is the distance between the center of data window k and the center of data window k+1
- an observed value for ⁇ in the k+1 window is measured Any number of well known methods may be used to measure ⁇ o(k+l)
- ⁇ d7 ⁇ + 1 ⁇ ⁇ dt ⁇ ⁇ ) + ⁇ ° ik + ⁇ ⁇ ⁇ p ⁇ k + ⁇ ⁇
- a and b are constants having values between 0 and 1
- the value chosen for a depends on the (i) rate of change of ⁇ , i e., magnitude of d ⁇ /dt, and (ii) the accuracy of the method used to measure ⁇ o(k+l) When the rate of change of ⁇ is high, and the accuracy of ⁇ o(k+l ) is high, the observed value of ⁇ is weighted more heavily then the predicted value, thus ⁇ is chosen to be large, e.g , 0.8 Alternatively, when the rate of change of ⁇ is low, and the accuracy of ⁇ o(k+l) is low, the predicted
- a plurality of possible values for the PSF are selected, and the largest value of the PSF which provides a non-negative result signal A(t) in a particular data window is used in the deconvolution method
- the FT of the signal and the PSF Prior to deconvolving the signal and the PSF, the FT of the signal and the PSF is taken, thus transforming the signal and the PSF from the time or position domain to the frequency domain
- the FT is taken using a fast Fourier transform (FFT) technique, e g , the Cooley-Tukey method
- FFT fast Fourier transform
- e g the Cooley-Tukey method
- the signal in the window is "zero-padded" at either the beginning or the end of the window in order to eliminate aliasing effects due to overlap of neighboring sample windows
- system noise refers to small random fluctuations in the signal resulting from fluctuations in components of the measurement device such as a radiation source, electronic circuitry, radiation detector, or any other like device-related source, or, from fluctuations in external environmental factors such as mechanical vibration, pick-up from 60-Hz electrical lines, temperature variations, or any other like environmental source
- n is characte ⁇ zed by a frequency of greater than 2/Np, where Np is the approximate number of data points making up a single sample zone or peak
- Np is the approximate number of data points making up a single sample zone or peak
- n is empirically estimated from prior knowledge of the application.
- One preferred method to determine a value for n is as follows.
- the FT of the random fluctuations in the signal is collected in a given data window, and the frequency at which the FT of the fluctuations does not appreciably change as a function of frequency is determined and the value of the FT of the fluctuations at that frequency is taken as the value of n.
- the preferred filter used to reduce the effects of the system noise on the deconvolved result is a Weiner filter , e.g., Fundamentals of Digital Image Processing, A K. Jain, pages 7 and 276, Prentice Hall, New Jersey ( 1989)
- a Weiner filter is defined by the function
- A(f) is the Fourier transform of the result signal
- D(f) is the Fourier transform of the signal
- P(f) is the Fourier transform of the point-spread-function
- P*(f) is the complex conjugate of P(f)
- n is the system noise Computer
- a computer can take the form of a generic microprocessor driven by appropriate software, a dedicated microprocessor using embedded firmware, or a customized digital signal processing circuit (DSP) which is dedicated to the specific data acquisition, analog-to-digital conversion, Fourier transformation, or filtering operation required by the signal processing method
- DSP digital signal processing circuit
- the computer comprises (i) a memory for storing a digitized representation of the signal, and (ii) a processor for carrying out the various steps of the signal processing method
- program storage device readable by a machine
- program storage device including a computer readable medium
- Computer readable media include magnetic diskettes, magnetic tapes, optical disks, Read Only Memory, Direct Access Storage Devices, and any other like medium
- the sequencing template was a pGEM 3Zf(+) plasmid
- the sampie was prepared according to standard protocols described in the document ABI Prism Dye Primer Cycle Sequencing Core Kit with AmpliTaq DNA Polymerase FS (ABD part number 402114, rev A, July 1995), with the exception that the nucleotide balance was slightly altered The altered nucleotide balance is shown in the table immediately below
- FIGS 6A and 6B show data from the early region of the signal both before (top) and after (bottom) deconvolution
- FIGS 7A and 7B show data from the middle region of the signal both before (top) and after (bottom) deconvolution
- FIGS 8A and 8B show data from the late region of the signal both before (top) and after (bottom) deconvolution
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Abstract
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT96942130T ATE215245T1 (de) | 1995-12-20 | 1996-12-02 | Aus analytischen trennungsprozessen resultierendes dekonvolutionsverfahren zur analyse von daten |
JP52283697A JP3555686B2 (ja) | 1995-12-20 | 1996-12-02 | 分析的分離プロセスから生じたデータの分析のための逆畳み込み方法 |
AU11284/97A AU702262B2 (en) | 1995-12-20 | 1996-12-02 | Deconvolution method for the analysis of data resulting from analytical separation processes |
CA002240539A CA2240539C (fr) | 1995-12-20 | 1996-12-02 | Procede de deconvolution servant a effectuer l'analyse de donnees obtenues au moyen de processus de separation analytique |
EP96942130A EP0868702B1 (fr) | 1995-12-20 | 1996-12-02 | Procede de deconvolution servant a effectuer l'analyse de donnees obtenues au moyen de processus de separation analytique |
DE69620232T DE69620232T2 (de) | 1995-12-20 | 1996-12-02 | Aus analytischen trennungsprozessen resultierendes dekonvolutionsverfahren zur analyse von daten |
Applications Claiming Priority (2)
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US08/579,204 | 1995-12-20 | ||
US08/579,204 US5748491A (en) | 1995-12-20 | 1995-12-20 | Deconvolution method for the analysis of data resulting from analytical separation processes |
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WO1997022942A1 true WO1997022942A1 (fr) | 1997-06-26 |
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PCT/US1996/019242 WO1997022942A1 (fr) | 1995-12-20 | 1996-12-02 | Procede de deconvolution servant a effectuer l'analyse de donnees obtenues au moyen de processus de separation analytique |
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US (1) | US5748491A (fr) |
EP (1) | EP0868702B1 (fr) |
JP (1) | JP3555686B2 (fr) |
AT (1) | ATE215245T1 (fr) |
AU (1) | AU702262B2 (fr) |
CA (1) | CA2240539C (fr) |
DE (1) | DE69620232T2 (fr) |
WO (1) | WO1997022942A1 (fr) |
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1995
- 1995-12-20 US US08/579,204 patent/US5748491A/en not_active Expired - Lifetime
-
1996
- 1996-12-02 EP EP96942130A patent/EP0868702B1/fr not_active Expired - Lifetime
- 1996-12-02 AU AU11284/97A patent/AU702262B2/en not_active Ceased
- 1996-12-02 WO PCT/US1996/019242 patent/WO1997022942A1/fr active IP Right Grant
- 1996-12-02 AT AT96942130T patent/ATE215245T1/de not_active IP Right Cessation
- 1996-12-02 CA CA002240539A patent/CA2240539C/fr not_active Expired - Fee Related
- 1996-12-02 DE DE69620232T patent/DE69620232T2/de not_active Expired - Lifetime
- 1996-12-02 JP JP52283697A patent/JP3555686B2/ja not_active Expired - Fee Related
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KONDO H ET AL: "ADAPTIVE IMAGE RESTORATION USING CONSTRAINED DECONVOLUTION", TRANSACTIONS OF THE INSTITUTE OF ELECTRONICS, INFORMATION AND COMMUNICATION ENGINEERS OF JAPAN, vol. E72, no. 11, 1 November 1989 (1989-11-01), pages 1243 - 1250, XP000086632 * |
MEHMET BILGEN ET AL: "RESTORATION OF NOISY IMAGES BLURRED BY A RANDOM POINT SPREAD FUNCTION", PROCEEDINGS OF THE INTERNATIONAL SYMPOSIUM ON CIRCUITS AND SYSTEMS, NEW ORLEANS, MAY 1 - 3, 1990, vol. 1 OF 4, 1 May 1990 (1990-05-01), INSTITUTE OF ELECTRICAL AND ELECTRONICS ENGINEERS, pages 759 - 762, XP000166934 * |
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US7593819B2 (en) | 2001-07-11 | 2009-09-22 | Applied Biosystems, Llc | Internal calibration standards for electrophoretic analyses |
US8268558B2 (en) | 2001-07-11 | 2012-09-18 | Applied Biosystems, Llc | Internal calibration standards for electrophoretic analyses |
JPWO2016120959A1 (ja) * | 2015-01-26 | 2017-06-29 | 株式会社島津製作所 | ノイズレベル推定方法、測定データ処理装置、及び測定データ処理用プログラム |
EP3252465A4 (fr) * | 2015-01-26 | 2018-01-17 | Shimadzu Corporation | Procédé d'estimation de niveau de bruit, dispositif de traitement de données de mesure et programme de traitement de données de mesure |
EP3260858A4 (fr) * | 2015-02-16 | 2017-12-27 | Shimadzu Corporation | Procédé d'estimation d'un niveau de bruit, dispositif de traitement de données de mesure et programme pour le traitement des données de mesure |
US11467140B2 (en) | 2017-11-15 | 2022-10-11 | Mecanique Analytique Inc. | Method and apparatus for analysing a gas chromatography elution peak |
Also Published As
Publication number | Publication date |
---|---|
US5748491A (en) | 1998-05-05 |
DE69620232T2 (de) | 2002-11-14 |
EP0868702A1 (fr) | 1998-10-07 |
AU702262B2 (en) | 1999-02-18 |
EP0868702B1 (fr) | 2002-03-27 |
CA2240539C (fr) | 2002-04-23 |
JPH11506858A (ja) | 1999-06-15 |
ATE215245T1 (de) | 2002-04-15 |
JP3555686B2 (ja) | 2004-08-18 |
CA2240539A1 (fr) | 1997-06-26 |
AU1128497A (en) | 1997-07-14 |
DE69620232D1 (de) | 2002-05-02 |
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